Reactive electrophiles generated by environmental agents and stresses can modify proteins and trigger adaptive cellular responses reflected by gene expression and protein functional changes. Cysteine thiol groups are thought to be key sites of important oxidative or covalent adduct modification, but the specific structural characteristics that cast proteins in sentinel roles are unknown. The overall objective of this project is to identify specific protein targets that serve as triggers for cellular stress responses. We hypothesize that specific structural features allow some alkylation sensitive proteins to act as "sensor-triggers" that readily react with electrophiles and that adduction alters critical protein-protein interactions, thus causing changes in kinase signaling and transcription factor regulation. We will use new proteomics liquid chromatography-tandem mass spectrometry (LC-MS-MS) tools and approaches to study the mechanisms by which protein modifications alter key protein-protein interactions that regulate stress responses. The specific aims of the proposed research are: 1) To identify protein classes, sequence motifs and domain structures that are targets for thiol-reactive electrophiles in human cells. Biotin-linked electrophiles will be used to combine affinity enrichment and new LC-MS-MS approaches to identify modified proteins and map the adducts to specific sequences. Different thiol-reactive electrophile chemistries will be used to establish the generality of the targets identified. Bioinformatics tools will be used to associate adducted sequences with specific domain structures and motifs. 2) To validate specific proteins as targets for thiol-reactive electrophiles in intact cells. Proteins identified as targets for biotin-linked electrophiles in Specific Aim 1 will be evaluated as targets for model electrophiles that correspond to their biotin-linked counterparts in cell models. 3) To characterize the effects of protein adduction on selected protein enzymatic activities and on selected protein-protein interactions in reconstituted systems in vitro. Quantitative proteomics methods will be applied to compare adduction with changes in target activities and protein-protein interactions in a parallel in vitro model. These studies will lay the groundwork for applying molecular target analysis to proteomes in toxicology.